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Am J Physiol Lung Cell Mol Physiol 295: L489-L496, 2008. First published June 27, 2008; doi:10.1152/ajplung.90282.2008
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EGF mediates calcium-activated chloride channel activation in the human bronchial epithelial cell line 16HBE14o–: involvement of tyrosine kinase p60c-src

Claudette Jeulin,1 Virginie Seltzer,1 Danielle Bailbé,2 Karine Andreau,1 and Francelyne Marano1

1Laboratoire de Cytophysiologie et Toxicologie Cellulaire and 2Laboratoire de Physiopathologie de la Nutrition, Centre National de la Recherche Scientifique-ESA 7059, Université Paris 7 Denis Diderot, Paris, France

Submitted 18 April 2008 ; accepted in final form 26 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Particulate atmospheric pollutants interact with the human airway epithelium, which releases cytokines, chemokines, and EGF receptor (EGFR) ligands leading to proinflammatory responses. There is little information concerning the short-term effects of EGFR activation by extracellular ligands on ionic regulation of airway surface lining fluids. We identified in the membrane of human epithelial bronchial cells (16HBE14o– line) an endogenous calcium- and voltage-dependent, outwardly rectifying small-conductance chloride channel (CACC), and we examined the effects of EGF on CACC activity. Ion channel currents were recorded with the patch-clamp technique. In cell-attached membrane patches, CACC were activated by exposure of the external surface of the cells to physiological concentrations of EGF without any change in cytosolic Ca2+ concentration ([Ca2+]i) and inhibited by tyrphostin AG-1478 (an inhibitor of EGFR that also blocks EGF-dependent Src family kinase activation). EGF activation of c-Src protein in 16HBE14o– cells was observed, and the signaling pathway elicited by EGFR was blocked by tyrphostin AG-1478. In excised inside-out membrane patches CACC were activated by exposure of the cytoplasmic face of the channels to the human recombinant Src(p60c-src) kinase with endogenous or exogenous ATP and inhibited by {lambda}-protein phosphatase. Secretion of EGFR ligands by epithelial airway cells exposed to pollutants would then elicit a rapid and direct ionic response of CACC mediated by EGFR activation via a Src kinase family-dependent signaling pathway.

calcium-activated chloride channels; patch-clamp technique; epidermal growth factor receptor; Src tyrosine kinase

THE AIRWAY EPITHELIUM IS the target of signaling events that serve to regulate ion secretion and water equilibrium of surface lining fluids and mucus production by goblet cells. Studies of epithelial airway cells in vitro have defined some of the molecular events induced by atmospheric pollutants. The epidermal growth factor (EGF) receptor (EGFR, c-erbB1) plays a pivotal role in maintenance and repair of airway epithelial tissues. After injury, the normal epithelium should respond, driven by ligands acting on EGFR or through transactivation of the receptor. It can be activated by asbestos fibers (34), cigarette smoke (26) and particulate matter (PM), leading to activation of the MAP kinase signaling cascade and cell proliferation (21) and to cytokine expression and secretion (33). In human bronchial epithelial cells (16HBE14o–), matter with an aerodynamic diameter <2.5 µm (PM2.5) and diesel exhaust particles upregulate the expression of amphiregulin (AR), an EGFR ligand that contributes to the secretion of granulocyte macrophage colony-stimulating factor (GM-CSF), leading to inflammation of the airways (4). The secretion of an EGFR ligand that produces GM-CSF release may therefore reflect an important mechanism for sustaining the proinflammatory response.

There is little information concerning the effects of extracellular ligands of EGFR on Cl– channels that participate in Cl– secretion from the apical membrane of airway epithelial cells. Ligand binding to receptor tyrosine kinases (RTKs) leads to receptor dimerization, kinase activation, and autophosphorylation on tyrosine residues. These phosphorylated tyrosines then serve as docking sites for the SH2 domains of a variety of signaling molecules including sarcoma virus tyrosine kinases (Src kinases). The precise role of Src kinases as signal transducers is under intensive investigation. Several plasma membrane ion channels are regulated by tyrosine phosphorylation (8). Activation of EGFR kinases has different effects on ion channels. In Chinese hamster ovary cells expressing epithelial sodium channel (ENac), EGF decreases ENac open probability (Po) (31). In T84 colonic epithelial cells, transactivation of EGFR by signaling pathway is coupled to inhibition of Ca2+-dependent Cl– secretion in response to Ca2+-mediated agonists (19). Src kinase has major effects on the cystic fibrosis transmembrane conductance regulator (CFTR) channel in the airway cell line calu-3 and in CFTR-transfected 3T3 fibroblasts (11). Src and EGFR kinases have opposing effects on volume-sensitive Cl– current in human atrial myocytes (9).

In the present study, using patch-clamp technique, we examined the effects of EGF on the activity of a Ca2+-activated chloride channel (CACC) in the membrane of 16HBE14o– cells. These defined cultured cells originate from human bronchial epithelial cells transformed by an origin-defective simian virus, SV40 (7). They express the CFTR channel (7), outwardly rectifying Cl– channels, and nonselective cation channels (15, 16), but they show no amiloride-sensitive Na+ conductance (16, 20). We identified in 16HBE14o– cells an endogenous calcium- and voltage-dependent outwardly rectifying small conductance Cl– channel, regulated by extracellular physiological concentrations of EGF without changing cytosolic Ca2+ concentration ([Ca2+]i) and activated by a Src kinase-dependent signaling pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. The cell line 16HBE14o– was a generous gift from Dr. D. C. Gruenert (University of Vermont, Burlington, VT). Cells were used after a limited number of seedings (n = 45). During this period, epinephrine stimulated apical Cl– secretion from the monolayer. The cells were grown on 35-mm-diameter Falco Primaria culture dishes that had been coated with 200 µl of 1/10 diluted extracellular matrix product (BTI, Biomatrix I, Clinisciences, Montrouge, France). The medium was Dulbecco's modified Eagle's medium (DMEM)-Ham's F-12 without HEPES (GIBCO-Invitrogen, Cergy-Pontoise, France) supplemented with Ultroser G (2%; Pall BioSepra, Cergy-St Christophe, France), 50 U/ml penicillin, and 50 µl/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.

Immunofluorescence. 16HBE14o– cells were grown for 3 days in a Lab-Tek II chamber glass slide system (Nalge Nunc, Naperville, IL) that had been coated with extracellular matrix product (BTI, Biomatrix I). Cells were washed in PBS and then fixed in methanol at –20°C. Primary antibodies were against human anti-EGFR and mouse monoclonal IgG1 (Upstate cell signaling solutions, Euromedex, Mundolsheim, France; 1/200). Secondary antibodies were against mouse red Alexa (F546, 1/500). Nuclei were counterstained with DAPI (2 µg/ml). Cells were photographed under a UV conventional fluorescence microscope.

Cytosolic [Ca2+] measurements. 16HBE14o– cells were grown on 24-mm-diameter coverslips that had been coated with extracellular matrix product (BTI, Biomatrix I) in 35-mm-diameter Falcon Primaria culture dishes as described above. After 4–6 days small cell clusters of 10–20 cells were loaded with 5 µM fura-2 acetoxymethyl ester (fura-2 AM, Molecular Probes, Leiden, The Netherlands) for 1 h at 37°C in a solution containing (in mM) 115 NaCl, 5 KCl, 24 NaHCO3–, 1 CaCl2, 1 MgCl2, and 5.5 glucose, with 5 mg/ml BSA. The coverslips were then placed in a 1-ml-volume perifusion chamber on the stage of an inverted fluorescent microscope (Nikon, Diaphot, Champigny-sur-Marne, France) and maintained at 37°C in a climate box (22). The chamber was perfused with a physiological solution that contained (in mM) 125 NaCl, 5.9 KCl, 1.28 CaCl2, 1.2 MgCl2, 2.8 glucose, and 25 mM HEPES (pH 7.4), with 1 mg/ml BSA via cannulas connected to a peristaltic pump. The microscope was equipped with a single quartz fiber illumination system and a (x40) fluor oil immersion objective. A selected area of one cell cluster was excited alternately at 340 and 380 nm every 2 s, and the fluorescence emitted at 510 nm was measured with a Photoscan II microfluorometer (Photon Technology International, Biotek Kontron, St-Quentin en Yvelines, France). Background fluorescence was recorded for both wavelengths in areas void of cell clusters, and this was subtracted from the measurements of fura-2 AM-loaded cells. The autofluorescence of the cells was similar to that of the background fluorescence estimated in the same measurement window. The measurement of successive 340-to-380 nm fluorescence ratios was taken to reflect [Ca2+]i.

Immunoblot analysis. Cells were grown to a density of 5 x 103 cells/cm2 on plastic tissue culture dishes coated with collagen (type I, from calf skin; Sigma). Serum-starved cells were treated with EGF (25 ng/ml) for between 15 min and 16 h. In some experiments, cells were incubated with tyrphostin AG-1478 (1 µM) for 1 h before the application of EGF. Experiments were terminated by the addition of PBS lysis buffer (100 µl for 106 cells) containing 0.1% Triton X-114, 1 mM orthovanadate, and protease inhibitor cocktail (Sigma). Cells were harvested by scraping (4°C for 15 min), and total protein extract was obtained by centrifugation at 12 000 g for 15 min at 4°C. Forty micrograms of total protein extracts was run on 8% SDS-PAGE. Immunodetection was performed with affinity-purified anti-c-Src family [c-Src (SRC2): sc-18, 1/500] and anti-p-Tyr (PY20: sc-508, 1/200) (both antibodies from Santa Cruz Biotechnology Europe). Horseradish peroxidase-conjugated goat anti-rabbit and rabbit anti-mouse (1/5,000, Dako, Trappes, France) were used as secondary antibodies, and chemiluminescence revelation was performed (Perkin Elmer Europe, Milan, Italy). To check that equal amounts of protein were blotted, nitrocellulose membranes were stripped (0.1 M NaCl, 0.1 M glycine, 0.2% SDS pH 3, for 20 min at room temperature) and probed with anti-{alpha}-tubulin (DM1A, 1/10,000; Sigma).

Experimental solutions and chemicals for electrophysiological experiments. To enhance the recording of Cl– channel currents during patch-clamp experiments, bath and pipette solutions were nominally free of monovalent cations. The bath solution contained (in mM) 145 NMDG-Cl–, 1.4 MgCl2, 1 CaCl2, and 10 N-tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), osmolality 300 mosmol/kgH2O. The patch pipettes were filled with a solution containing (in mM) 145 NMDG-Cl–, 2 MgCl2, 2 CaCl2, and 10 TES, 300 mosmol/kgH2O. The low-NMDG-Cl– bath solution consisted of (in mM) 30 NMDG-Cl–, 1.4 MgCl2, 1 CaCl2, and 10 TES, 300 mosmol/kgH2O adjusted with sucrose. The pH of all solutions was 7.4. Solutions were prepared with Ultra pure water (Milli-Q system, Millipore, St-Quentin en Yvelines, France). The free Ca2+ concentration in the bath solution was adjusted with Ca2+ and EGTA (Sigma, St Quentin Fallavier, France).

The human recombinant tyrosine kinase p60c-src (Upstate Biotechnology, Lake Placid, NY) was diluted in bath solution, ~6–12 U/ml. The viral serine/threonine/tyrosine phosphatase, {lambda}-protein phosphatase ({lambda}-PP, Sigma) was diluted at 200 U/ml in (mM) 2 MnCl2, 50 Tris·HCl, 0.1 Na2-EDTA, and 5 dithiothreitol, with 0.01% Brij 35, pH 7.5.

Electrophysiology. Ion channel currents were recorded with the patch-clamp technique in cell-attached and inside-out membrane patch configurations on the surface of membranes obtained from the periphery of small clusters of nonconfluent cells grown on extracellular matrix. This method avoided enzyme treatment. The bath solution surrounding cells or excised membrane patches was perfused with a gravity-driven, multibarrel perfusion system (7 reservoirs) placed within 100 µm of the pipette and delivering 34 µl/min. Solution changes were achieved within 10 s by manual switching between reservoirs. Experiments were performed at room temperature (21–23°C). Recording pipettes were pulled in two stages from borosilicate glass capillary tubes (GC 150-7.5, Clark Electromedical Instruments, Reading, UK). Pipettes were coated with two layers of Sylgard 184 (Dow Corning Europe, Brussels, Belgium) and fire polished and had a resistance of 15–20 M{Omega} when filled with the pipette solution. The reference Ag-AgCl electrode was connected to the bath via an NMDG-Cl– agar bridge. Single-channel currents were recorded with an Axopatch 200B amplifier (Axon Instruments, Dipsi Industrie, Chatillon-sous-Bagneux, France), filtered with a four-pole Bessel filter at 1 kHz and recorded on digital audiotape (DAT DTR, 1205, Biologic, Claix, France). Current recordings were converted with an analog-to-digital interface (Card Lab Master, DMA 100 OEM, Biologic) coupled to a PC-compatible computer running appropriate software (pCLAMP, v. 6, Axon Instruments, Foster City, CA). Currents were digitized at 20 kHz. Recording sequences (30 or 60 s or several minutes) were chosen on replay of DAT cassettes and then transferred to storage media (ZIP disks, Iomega, Ropy, UT) or to a printer (Dash IV model XL, Astro-Med, Trappes, France) for long sequences.

Data analysis. Single-channel data were analyzed with pCLAMP software (v. 6). Channels were identified and characterized according to their ionic selectivity with respect to a NMDG-Cl– concentration gradient (30 mM in the bath vs. 145 mM NMDG-Cl– in the pipette) and their single-channel conductance. Unitary current reversal potential and conductance values were estimated from the linear portion of current-voltage (I-V) relationships. Channel amplitude was calculated from Gaussian fits to amplitude/distribution histograms constructed from single-channel recordings. The probability of a channel being open (Po) was measured during 30 s of stable and representative recordings. To calculate Po, digitized single-channel data were subjected to event detection (pCLAMP). Po was calculated as the fraction of the specified recording time spent by the channel in the open state.

Statistics. Results are reported as means ± SE. Significance was tested at P = 0.05 with the Kruskal-Wallis nonparametric test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Single CACC currents in human bronchial 16HBE14o– cells. In 80 cell-attached patches (Fig. 1A) and 44 inside-out patches, we recorded single-channel currents characterized by a strong outward rectification and a high rate of channel activation with depolarization.


Figure 1
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  		Fig. 1. Single Cl– channel currents in human bronchial 16HBE14o– cells. A: voltage stimulates single-channel currents in cell-attached patches on the apical membrane of cells. Single-channel currents were recorded at different membrane potentials (Vm, right) in symmetric 145 mM N-methyl-D-glucamine (NMDG)/Cl– solution. In control experiments, the bath solution contained 10–3 M Ca2+. c, Closed state of the channel; o, open state of the channel. Channel activation increased with depolarization and channel activity was observed at Vm of 0 mV (n = 80). B: channel open probability (Po) as a function of Vm in inside-out patches from apical membrane of cells. Measurements of Po (n = 3 patches) during 30-s periods of stable and representative channel activity at different holding potentials are shown. Values are means ± SE. C: effects of intracellular Ca2+ concentration ([Ca2+]) on Cl– channel activity: example of a current recording from an inside-out patch, 2 min after excision, in symmetric 145 mM NMDG/Cl– solution. The bath solution [Ca2+] was changed from 10–3 M to 10–8 M as indicated (right). Data were recorded at Vm of +70 mV. These observations were reproduced in 10 different inside-out patches. D: current-voltage (I-V) relationships of the Cl– channel in cell-attached and inside-out patches from the apical membrane of cells. Unitary I-V relationships were obtained from 6 cell-attached patches ({blacktriangleup}) and from 6 inside-out patches (bullet) of the apical membrane of 16HBE14o– cells, containing 1 channel at hyperpolarizing voltages, under symmetric 145 mM NMDG/Cl– solutions (control bath solution). Single-channel currents were also recorded with asymmetric Cl– conditions with 30 mM ({circ}, n = 3) NMDG/Cl– in the bath solution (means ± SE). Dotted, black, and gray lines are regression lines corresponding to cell-attached, inside-out patches in symmetric and asymmetric conditions, respectively.

 
The unitary I-V relationship obtained from these channels in cell-attached patches in symmetric NMDG/Cl– solutions was rectified over the range –80 to +100 mV (Fig. 1D). The slight outward rectification was assessed from the slopes of I-V relations. The mean conductance of the channels was 7.5 ± 0.3 pS at +70 mV and 1.6 ± 0.9 pS at –70 mV. The reversal potential was –60 mV. The unitary I-V relationships from cell-attached and inside-out patches in symmetric 145 mM NMDG/Cl– solutions show the same outwardly rectifying small-conductance channels. In inside-out membrane patches the mean conductance was 6.2 ± 0.8 pS at +60 mV and 3 ± 0.7 pS at –60 mV and the reversal potential was 0 mV (Fig. 1D). When intracellular NMDG/Cl– was reduced to 30 mM (Fig. 1D), the reversal potential was –40 mV ± 5 mV, which is close to the value of –40.2 mV predicted by the Goldman-Hodgkin-Katz voltage equation for a perfectly anion-selective channel at 25°C. These experimental conditions favor the recording of anionic channel currents, and separate experiments showed that these channels were more permeant to I– and Br– than Cl– (data not shown). This selectivity was not different from those of CACC described in human nasal epithelial cells (17). We conclude that these recordings represent an anion-selective channel.

In cell-attached membrane patches, Cl– channel activity was not sustained but declined and disappeared within several minutes of the onset of recording. In inside-out membrane patches channel activity also ran down within 1–5 min after excision. Before rundown, in both cell-attached and inside-out patches Cl– channel activity increased with depolarization (Fig. 1A). In inside-out patches Po increased from 0.03 ± 0.02 at –90 mV to 0.58 ± 0.11 at +90 mV (n = 3, Fig. 1B).

Figure 1C illustrates the internal Ca2+ dependence of channel activity. Reducing [Ca2+] in the bath solution from 10–3 to 10–7 M decreased Po from 0.48 ± 0.05 to 0.06 ± 0.03 (n = 5, P < 0.05), and channel activity ceased completely at 10–8 M [Ca2+]. This effect was rapidly reversible, and recovery of channel activity was obtained by increasing [Ca2+] in the bath from 10–8 to 10–3 M (n = 10). Other experiments (not shown, n = 3) demonstrated that these voltage- and Ca2+-activated chloride channels (CACC) could also be activated by Ca2+/CaM kinase II (0.10 µg/ml) in the presence of 10–5 M Ca2+, CaM (20 µg/ml), and ATP (500 µM). This effect was blocked by the application of 3 nM CaM kinase II inhibitory peptide (281-309). These observations are similar to those reported for CACC in the apical membranes of nonciliated human nasal epithelial cells (17).

In summary, these results (Fig. 1) demonstrate outwardly rectified, voltage- and calcium-activated Cl– channels in the membranes of human bronchial 16HBE14o– cells.

The effect of EGF on CACC. 16HBE14o– cells express EGFR on the cytoplasmic membrane (Fig. 2A), and the direct exposure of intact cells to EGF (20–200 ng/ml) elicited, after a variable delay (5–70 s), a marked increase in channel Po recorded in cell-attached patches (Fig. 2B). The addition of EGF (20–200 ng/ml) to the bath solution increased Po significantly (P < 0.05), in a dose-response relationship, from 0.01 ± 0.004 to 0.25 ± 0.04 at membrane potential (Vm) = +100 mV (Fig. 2C). Figure 2D illustrates the effect of EGF concentrations on CACC channel activity recorded in cell-attached patches. Increasing EGF concentration in the control bath solution from 50 to 100 and 150 ng/ml increased Po from 0.02 ± 0.05 to 0.20 ± 0.07 and 0.33 ± 0.07, respectively. The addition of tyrphostin AG-1478 (0.8 µM), an inhibitor of EGFR that also blocks EGF-dependent Src-family kinase activity, decreased Po from 0.43 ± 0.05 to 0.10 ± 0.06 (n = 5). In each of these experiments the identity of CACC was confirmed by recording their voltage dependence in cell-attached membrane patches and I-V relations.


Figure 2
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  		Fig. 2. Effect of EGF on calcium-activated Cl– channels (CACC). A: localization of EGF receptors (EGFR) in the membrane of polarized 16HBE14o– cells by indirect immunofluorescence microscopy. Cells were labeled with primary human anti-EGFR antibodies. Bars, 10 µm. B: dose-response relationship between EGF concentrations and the effect of EGF on channel activity at Vm of +100 mV. Current recording from a cell-attached patch in control bath solution containing 10–3 M Ca2+: the loss/rundown of Cl– channel activity 3 min after seal, before (a) and after (5–70 s) exposure to EGF solvent (b) or 20 (c), 50 (d), 100 (e), or 150 (f) ng/ml EGF. Experiments were reproduced: EGF solvent (n = 10), 20 ng/ml EGF (n = 4), 50 ng/ml EGF (n = 20), 100 ng/ml EGF (n = 16), and 150, 200, and 300 ng/ml EGF (n = 4 for each concentration). C: effects of extracellular [EGF] (10–200 ng/ml) on Cl– channel activity: Po at Vm of +100 mV recorded in 3 different cell-attached patches (means ± SE; *significance P < 0.05). D: effects on channel activity of exposure of intact cells to EGF and tyrphostin AG-478. Traces a–c represent parts of an otherwise continuous (several minutes) recording of single-channel current activity in cell-attached patches. Data were recorded at Vm of +80 mV and –80 mV. a: Current recording from a cell-attached patch in symmetric 145 mM NMDG/Cl– bath solution containing 10–3 M Ca2+ that showed the loss/rundown of Cl– channel activity 4 min after seal; Cl– channels were not activated 20 s after exposure to EGF solvent (P = 0). Cl– channels were activated 30 s after exposure to 50 ng/ml EGF (P = 0.02 ± 0.05) and 50 s after exposure to 100 ng/ml EGF (P = 0.20 ± 0.07). b: Cl– channels were activated 50 s after exposure to 150 ng/ml EGF (Po = 0.33 ± 0.07) and 120 s after exposure to 150 ng/ml EGF (Po = 0.43 ± 0.05). c: Addition of 0.4 and 0.8 µM tyrphostin AG-478 decreased channel activity (Po = 0.10 ± 0.06). These observations were reproduced in 5 patches.

 
Figure 3 shows that EGF had no effect on [Ca2+]i in 16HBE14o– cells while both UTP and ATP, acting via apical membrane P2y2 receptors, stimulated an increase in [Ca2+]i. This result was reproduced in three experiments. External ATP and UTP cause a transient increase in [Ca2+]i and Isc (stimulation of Cl– secretion) when added to the apical surface of 16HBE14o– cell monolayer and nontransformed human bronchial epithelial cells (2, 32). These experiments suggest that these voltage- and calcium-activated Cl– channels were activated by EGF (Fig. 2, B–D) by a mechanism that did not involve an increase in [Ca2+]i (Fig. 3).


Figure 3
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  		Fig. 3. Effects of EGF on cytosolic [Ca2+] ([Ca2+]i) as measured in a cluster of 10–20 cells. [Ca2+]i of 16HBE14o– cells was measured [fluorescence (F) ratio of 340 to 380 nm] with fura-2 AM with the protocol described in MATERIALS AND METHODS. The fura-2-loaded cells were perifused with a Krebs-Ringer bicarbonate (KRB) solution containing no additives (Basal), EGF solvent, 100 ng/ml EGF, Basal, 135 µM uridine triphosphate (UTP), Basal, 200 µM magnesium adenosine triphosphate (ATP), Basal. These observations were reproduced in 3 experiments.

 
In excised membrane patches the direct exposure of the cytoplasmic face of CACC to Src tyrosine kinase (p60c-src) increased Po (Fig. 4A) in a bath solution containing 10–3 M Ca2+. This effect was reproducible depending on the quantity of Src kinase and exogenous ATP in the control bath solution. In some experiments, the direct activation of CACC by Src kinase was rapid (10 s, Fig. 4A). In other experiments, the addition of exogenous ATP (500 µM) in the bath solution, after a first application of 12 U of Src kinase, increased channel activity after 9-min exposure to the mixture (Fig. 4D). The direct activation of CACC by Src kinase was reversed by the application of 200 U of {lambda}-PP (Fig. 4C). In the same patch membrane, channel activity could be regained with a second application of Src kinase, and this effect was rapidly reversible by a second application of {lambda}-PP. In each of these experiments, the identity of CACC was confirmed by recording their voltage dependence in inside-out membrane patches (Fig. 4B) with controlled I-V curve, channel conductance, and calcium dependence (by decreasing [Ca2+] in the bath solution from 10–3 M to 10–8 M, data not shown), easier to use in the patch-clamp technique than CACC inhibitor. Activation by Src kinase and calcium was not additive in the inside-out configuration.


Figure 4
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  		Fig. 4. Effects on channel activity of exposure of the cytoplasmic face of 16HBE14o– cells to Src (p60c-src) tyrosine kinase. A–C represent parts of an otherwise continuous recording of single-channel current activity in an inside-out patch of the apical membrane of cells. A: current recording from an inside-out patch in symmetric 145 mM NMDG/Cl– bath solution containing 10–3 M Ca2+ that showed the loss/rundown of Cl– channel activity 5 min after seal. Cl– channels were activated 8 s after exposure to 6 U of Src (p60c-src) kinase (>1 channel, and these observations were reproduced in 3 experiments). B: currents during a voltage-clamp ramp between +80 and –100 mV reveal the voltage dependence of CACC. C: effects on channel activity of 25-s exposure of 200 U of {lambda}-protein phosphatase ({lambda}-PP). Decreased channel activity was observed (only 1 or 2 open channels). These observations were reproduced in 3 patches. D: current recording from another inside-out patch, in the same control bath solution, which showed loss/rundown of Cl– channel activity 3 min after seal. Cl– channels were activated 9 min after exposure to 12 U of Src (p60c-src) tyrosine kinase and 500 µM ATP. These observations were reproduced in 3 experiments.

 
EGF-dependent activation of Src family kinases was shown in 16HBE14o– cells (Fig. 5). Cells were treated with a physiological concentration of EGF (25 ng/ml) for between 15 min and 16 h, and Western blots were performed with affinity-purified c-Src-family kinase antibody detecting phosphorylated and nonphosphorylated forms of Src kinases. Activation of Src kinase protein was rapid (<15 min) and persistent up to 16 h during EGF treatment. These increases were not observed when the cells had been treated with 1 µM tyrphostin AG-1478 for 1 h before EGF activation maximal at 16 h. Recombinant Src kinase was used as a control for the detection of phosphorylated and nonphosphorylated proteins in the 16HBE14o– cells. The phosphorylated form of Src kinase migrated slowly than the nonphosphorylated Src protein (Fig. 5). It has been reported that phosphorylated IRAK-4 protein from the IL-1 receptor signaling complex migrates more slowly in SDS-PAGE than the nonphosphorylated form, as did recombinant protein autophosphorylated in vitro (5).


Figure 5
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  		Fig. 5. Western blot analysis of EGF-induced Src-family kinase activation in 16HBE14o– cells. A: time course of EGF-induced Src-kinase phosphorylation. Anti-Src antibodies detected phosphorylated (P; active) and nonphosphorylated (inactive) forms of Src protein. Active Src kinase expression increased during EGF stimulation. Five units of human recombinant Src kinase protein Src-p 60c-src (rec) and anti-PY were used to discriminate Src phosphorylated and nonphosphorylated forms. B: tyrphostin AG-1478 effect on EGF induced Src-kinase phosphorylation. Phosphorylated Src family kinase expression was inhibited by 1-h tyrphostin AG-1478 treatment (1 µM) on 16-h EGF stimulation. Blotting with anti-{alpha}-tubulin showed that an equal amount of protein was probed. All experiments were performed 3 times, and representative data are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
EGF activation of endogenous CACC in 16HBE14o– cells via Src protein tyrosine activation. We have identified and characterized CACC in the membrane of human bronchial epithelial 16HBE14o– cells. In these cells, single CACC were outwardly rectified and Ca2+/CaM kinase II- and voltage dependent. Similar CACC have been reported in nonciliated human nasal epithelial cells (17).

Here, using the patch-clamp technique, we show that CACC were activated by direct exposure of the external surface of the intact 16HBE14o– cell membrane to EGF, a ligand of the EGFR. External application of EGF mediated CACC activation without change in intracellular [Ca2+], and several observations link this with Src protein tyrosine activation. First, the effect of EGF in intact cells was inhibited by tyrphostin AG-1478, which also blocks EGF-dependent Src tyrosine kinase activation. Second, in excised membrane patches CACC were activated by the direct application of recombinant Src (p60c-src) tyrosine kinase alone or with exogenous ATP. Third, the effect of Src tyrosine kinase was reversed by the addition of {lambda}-PP. Fourth, extracellular physiological concentrations of EGF increased phosphorylated forms of Src kinase in 16HBE14o– cells expressing endogenous EGFR. Activation was seen at 15 min and was maximal and persistent up to 16 h. Tyrphostin AG-1478 inhibited maximal Src kinase activation. Finally, we took advantage of the appropriated inside-out excised membrane patch technique to study the action of Src kinase on CACC. Indeed, results using Src kinase applied on the cytoplasmic face of the channel suggest a direct effect of Src kinase on CACC activation.

Extracellular EGFR activation by ligands and intracellular EGFR transactivation might have different effects on Cl– channels. EGFR transactivation in monolayer T84 colonic epithelial cells is coupled to inhibition of Ca2+-dependent Cl– secretion (Isc) in response to Ca2+-mediated agonists. This pathway constitutes an antisecretory mechanism by which carbachol-stimulated Cl– secretion is limited (19). In future investigation on airway cells, activation of P2y2 receptors by extracellular ATP or UTP, increasing intracellular calcium and Po of CACC, could be coupled to tyrphostin AG-1478 inhibition of EGFR in a cell-attached configuration of 16HBE14o– cells. However, interactive regulation of both types of receptors would be easier to study with Isc Cl– current on confluent monolayer. Furthermore, EGFR phosphorylation, transactivation, and trafficking and stability of the receptor complex were different from EGFR activation by ligands or under oxidative stress in A549 cells (18). EGF peptides increased and tyrphostin B46 inhibited the volume-sensitive outwardly rectifying (VSOR) Cl– current in murine mammary cells (1). Activation of c-Src tyrosine kinases with the Src activator peptide EPQ-(pY)EEIPI increased volume-sensitive Cl– current (ICl,vol) in nonpigmented ciliary epithelial cells (29). ICl,vol are regulated by the balance between protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activity in human atrial myocytes (9) and in rabbit ventricle myocytes (25). The human recombinant tyrosine kinase p60c-src increased and {lambda}-PP decreased CFTR currents in the airway cell line calu-3 and in CFTR-transfected 3T3 fibroblasts (11). In avian osteoclasts a novel Cl– channel, p62, has an affinity for both Src kinase SH2 and SH3 domains (10). In the present study, regulation of CACC by Src tyrosine activation is a mechanism associated with physiological stimulation.

This is the first time that c-Src kinase activation has been detected after external physiological EGF concentration treatment of airway epithelial cells (expressing endogenous EGFR) with anti-Src family antibodies (SRC2, Santa Cruz Biotechnology). These results can be compared with those found in NIH3T3 fibroblast cells, in which EGF-induced Src kinase activation was rapid (<1 min), persistent, and maximal up to 16 h only in cells overexpressing EGFR. High levels of EGFR expressed in cells showed more rapid and elevated Src family kinase activation (23).

The protooncogene Src was the first gene to be identified that is potentially capable of inducing cell transformation. The proteins of this group range from 52 to 62 kDa and comprise six distinct functional domains. The kinase domain may bind substrates after phosphorylation, thus promoting phosphorylation of other sequences of one or several neighboring substrate molecules (30). We postulate that activation of c-Src kinase by EGF induces activation of the CACC protein located in the membrane of 16HBE14o– cells. The CACC protein expressed in these cells has not yet been cloned. The CACC described here is endogenous to the cytoplasmic membrane of 16HBE14o– cells. It is different from hCLCA-2, which is expressed in human lung and trachea (13), because although antibodies against hCLCA2 stained the cytoplasm of 16HBE14o– cells it was not localized to the cytoplasmic membrane (data not shown). The CLCA proteins may in fact be Cl– channel regulators rather than channels in their own right.

Role of CACC in response of airway epithelia to ligands of EGFR. The relation between EGFR and CACC in airway epithelia is important in the reaction of the airway to stress (chemical stress, airborne PM, or cigarette smoke). Secretion of EGFR ligands may contribute to the persistent proinflammatory response as well as to the repair process and airway remodeling events. 16HBE14o– cells express EGFR and secrete ligands such as EGF, transforming growth factor (TGF)-{alpha}, and AR (24). Fine PM induces AR secretion by human bronchial epithelial cells and 16HBE14o– cells (4). AR gene was the most overexpressed (mRNA expression and secretion) by organic extract or native PM. AR is an EGFR ligand that contributes to GM-CSF release and the inflammatory response of airway cells to PM (26–28). Mechanisms of secretion of EGFR ligands by cells exposed to pollutants were under investigation. The three ligands (EGF, TGF-{alpha}, and AR) bound the same homodimeric receptor EGFR (c-erbB1). It could be a crucial actor in bronchial remodeling observed in the airway of chronically particle-exposed subjects and in asthmatic patients whose lungs overexpress EGFR. Furthermore, in human bronchial epithelial cells diesel exhaust particles induce activation of Stat3 (phosphorylation and nuclear translocation) through a process that includes the generation of reactive oxygen species and requires EGFR and Src (6). The secretion of EGFR ligands might rapidly activate CACC via Src signaling pathway and thus induce Cl– secretion at the apical pole as one of the first reactions of airway epithelia to stress. CACC also play a direct role in mucus production by goblet cells (14), and c-Src is a central element in the pathway connecting the CFTR channel with the overexpression of mucins (12). EGFR kinase inhibitors and CACC inhibitors have the potential to combat mucus overproduction (3). CACC may be a key signaling member that can be targeted with pharmacological interventions to treat mucus hypersecretion. Excessive opening of CACC might induce gradual cellular responses related to the reversibility, including changes in K+-Na+-2Cl– cotransporter activity, outwardly rectifying Cl– channel (ORCC) and CFTR channel activities, cell volume regulation, membrane polarization, activation of transcription factors required for the expression of genes related to production of cytokines and chemokines, apoptosis and necrosis, and thus sustained inflammation in the airways.


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This research was supported by grants 2005–2008 from Université Paris VII, Denis Diderot, EA DRED 1553.


   ACKNOWLEDGMENTS
 
We thank Annie Jaeger for technical assistance. We also thank Dr. Ian Findlay, Centre National de la Recherche Scientifique UMR 6542, Faculté des Sciences, Université François Rabelais, Tours, and Jacques Teulon, Centre National de la Recherche Scientifique, Centre de recherche Biomédicale des Cordeliers, Université Paris 5 René Descartes, Paris, for help, suggestions, and fruitful discussion of the manuscript.

C. Jeulin conceived the study and carried out the electrophysiological experiments. V. Seltzer carried out the electrophoretic studies, D. Bailbé and C. Jeulin carried out the cytosolic [Ca2+] measurements, K. Andreau had discussions concerning the study, and F. Marano participated in the final version of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Jeulin, Laboratoire de Cytophysiologie et Toxicologie Cellulaire, case courrier 7073, 3ème étage, T53-54, Université Paris 7 Denis Diderot, 2 Place Jussieu, 75251 Paris Cedex 05, France (e-mail: claudette.jeulin{at}univ-paris-diderot.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 RESULTS
 DISCUSSION
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